The traditional method for synthesis of proteins is to produce them in a laboratory or isolate them from natural sources. One major disadvantage of this process is that the desired protein is generated in small amounts and the task is laborious. With technological advances, especially in molecular biology, several important tools for protein synthesis have now become available. One such tool is the deployment of a host system such as a bacterium into which the gene encoding the protein of interest is cloned and culturing of the host system yields the protein of interest in large quantities.
While such a system marks a significant advance over traditional methods of protein production, this technique suffers from the disadvantage that the protein produced by the host requires extensive downstream processing of the cell lysate, which is time consuming and expensive. Further, such processing could cause denaturation of the protein or cause loss of activity of the protein isolated which defeats the idea of obtaining protein in active form.
Accordingly, one of the strategies employed in protein purification is to prepare a fusion protein i.e. a protein of interest fused to another protein. The “other” protein in the fusion protein may be a purification “tag” or a purification region. The purification “tag” or region is a protein, which although fused to the protein of interest enables the protein of interest to be purified to near homogeneity in a one step purification procedure using a resin with affinity for the tag. When the cell lysate is passed over a column, the tag binds to the resin column and along with it retains the protein of interest. All other proteins from the lysate do not bind and are eliminated in the flow-through of the column or the washes. The column-bound proteins are then released by passing a solution containing a small molecular weight competitor for the affinity tag that displaces the fusion protein.
The Glutathione-5-transferase (GST) fusion protein expression system is a popular system used for high level expression and subsequent purification of proteins in Escherichia coli. GST fusion proteins may be eluted competitively by using reduced glutathione without affecting the antigenicity and functional activity of recombinant proteins.
After a chimeric molecule (fusion protein) has been isolated, it is necessary to remove the “tag” prior to the use of the expressed protein of interest. “Tag” removal is typically achieved by use of specific proteases such as thrombin or factor Xa.
Although these proteases are considered specific for cleavage at the inserted cleavage site, proteolysis may frequently occur at other site(s) in the protein of interest. Additionally, relatively extended incubation times (4-16 h) are required for efficient cleavage by the two proteases listed above. As a result, there could be loss in amount of proteins obtained or labile proteins may lose their activity during such prolonged incubation periods. Further, in the GST-fusion protein system, the protease used for removal of GST-tag remains as a contaminant in the protein preparation.
To overcome the said problems in the art, it is desirable to provide an expression system comprising a cleavage site which is rare in naturally occurring proteins and which system can yield a cleaved product in a relatively short period. It is also desirable to provide an expression system that allows removal of the protease after cleavage of the “Tag”.